Humanized anti-ca ix antibodies and methods of their use

Abstract

A humanized antibody specifically recognizing proteoglycan domain of human CA IX, and to therapeutic and diagnostic methods utilizing this antibody is disclosed. The methods relate in particular to treatment or diagnosis of cancers selected from squamous cell carcinoma, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, hodgkin's lymphoma, non-hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, mesothelioma, and head and neck cancer.

1 . A humanized antibody specifically recognizing the proteoglycan domain of human CA IX containing a heavy chain variable region sequence comprising CDR sequences identical to or differing in 1 or 2 amino acids from the sequences GFTFNTNAMH (SEQ ID NO. 1), and RIRSKSNNYTTYYADSVKD (SEQ ID NO. 2), and VCGSWFAY (SEQ ID NO. 3); and a light chain variable region sequence comprising the CDR sequences identical to or differing in 1 or 2 amino acids from the following sequences: KSSQSLLNSSNQKNYLA (SEQ ID NO. 4), and FTSTRQS (SEQ ID NO. 5), and QQHYSIPLT (SEQ ID NO. 6). 
     
     
         2 . The humanized antibody according to  claim 1 , comprising at least one variable region selected from the group consisting of:
 a heavy chain variable region comprising or having the sequence:   
       
         
           
                 
               
                   (SEQ ID NO. 7) 
                 
                   X 32 VQLVESGGGX 33 VQPGX 34 SLX 35 LSCAAS GFTFNTNAMH WVRQAX 36 G 
                 
                   X 37 GLEWVX 38   RIRSKSNNYTTYYADSVKD RFTISRDX 39 SKX 40 TX 41 YLQ 
                 
                   X 42 NSLX 43 X 44 EDTAVYYC VCGSWFAY WGQGTX 45 VTVSS  
                 
             
                
                
                
                
               
            
           
         
          wherein
 X 32 =E or Q 
 X 33 =L or V 
 X 34 =G or R 
 X 35 =K or R 
 X 36 =S or P 
 X 37 =K or R 
 X 38 =A or G 
 X 39 =D or N 
 X 40 =N or S 
 X 41 =A or L 
 X 42 =M or V 
 X 43 =K or R 
 X 44 =T or A 
 X 45 =L or T; and 
 
         a light chain variable region comprising or having the sequence: 
       
       
         
           
                 
               
                   (SEQ ID NO. 8) 
                 
                   DX 46 X 47 MTQSPDSLAVSLGERX 48 TINC KSSQSLLNSSNQKNYLA WX 49 Q 
                 
                   QKPGQX 50 PX 51 X 52 X 53 IY FTSTRQS GVPDRFX 54 GSGSGTDFTLTIX 55 SL 
                 
                   QAEDVAVYX 56 C QQHYSIPLT FGQGTX 57 X 58 EIK 
                 
             
                
                
                
                
               
            
           
         
         
           X 46 =V or I 
           X 47 =V or Q 
           X 48 =V or A 
           X 49 =Y or F 
           X 50 =S or P 
           X 51 =K or N 
           X 52 =L or V 
           X 53 =L or V 
           X 54 =S or T 
           X 55 =S or N 
           X 56 =Y or F 
           X 57 =K or Q 
           X 58 =L or V. 
         
       
     
     
         3 . The humanized antibody according to  claim 2 , comprising at least one variable region selected from the group consisting of:
 a) a heavy chain variable region amino acid sequence comprising or having the sequences selected from the group consisting of   
       
         
           
                 
               
                   (SEQ ID NO. 9) 
                 
                   EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMH WVRQASGKGLEWVG 
                 
                     RIRSKSNNYTTYYADSVKD RFTISRDDSKNTAYLQMNSLKTEDTAVYYC 
                 
                     VCGSWFAY WGQGTLVTVSS, 
                 
                     
                 
                   (SEQ ID NO. 10) 
                 
                   EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMH WVRQASGKGLEWVG 
                 
                     RIRSKSNNYTTYYADSVKD RFTISRDDSKSTAYLQMNSLKTEDTAVYYC 
                 
                     VCGSWFAY WGQGTLVTVSS, 
                 
                     
                 
                   (SEQ ID NO. 11) 
                 
                   QVQLVESGGGVVQPGGSLRLSCAAS GFTFNTNAMH WVRQAPGRGLEWVA 
                 
                     RIRSKSNNYTTYYADSVKD RFTISRDNSKNTLYLQVNSLRAEDTAVYYC 
                 
                     VCGSWFAY WGQGTLVTVSS, 
                 
                     
                 
                   (SEQ ID NO. 12) 
                 
                   EVQLVESGGGVVQPGRSLRLSCAAS GFTFNTNAMH WVRQAPGKGLEWVA 
                 
                     RIRSKSNNYTTYYADSVKD RFTISRDNSKNTLYLQMNSLRAEDTAVYYC 
                 
                     VCGSWFAY WGQGTLVTVSS, 
                 
                   and 
                 
                     
                 
                   (SEQ ID NO. 13) 
                 
                   EVQLVESGGGLVQPGGSLKLSCAAS GFTFNTNAMH WVRQASGKGLEWVG 
                 
                     RIRSKSNNYTTYYADSVKD RFTISRDDSKNTAYLQMNSLKTEDTAVYYC 
                 
                     VCGSWFAY WGQGTTVTVSS; 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
          and 
         b) a light chain variable region amino acid sequence comprising or having the sequences selected from the group consisting of 
       
       
         
           
                 
               
                   (SEQ ID NO. 14) 
                 
                   DVVMTQSPDSLAVSLGERVTINC KSSQSLLNSSNQKNYLA WYQQKPGQS 
                 
                   PKLLIY FTSTRQS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQHY   
                 
                     SIPLT FGQGTKLEIK, 
                 
                     
                 
                   (SEQ ID NO. 15) 
                 
                   DIVMTQSPDSLAVSLGERATINC KSSQSLLNSSNQKNYLA WFQQKPGQP 
                 
                   PNLVIY FTSTRQS GVPDRFSGSGSGTDFTLTINSLQAEDVAVYFC QQHY   
                 
                     SIPLT FGQGTQVEIK, 
                 
                     
                 
                   (SEQ ID NO. 16) 
                 
                   DIQMTQSPDSLAVSLGERATINC KSSQSLLNSSNQKNYLA WYQQKPGQP 
                 
                   PKLLI YFTSTRQS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYFC QQHY   
                 
                     SIPLT FGQGTKVEIK, 
                 
                     
                 
                   (SEQ ID NO. 17) 
                 
                   DIVMTQSPDSLAVSLGERATINC KSSQSLLNSSNQKNYLA WFQQKPGQP 
                 
                   PKVLIY FTSTRQS GVPDRFTGSGSGTDFTLTISSLQAEDVAVYYC QQHY   
                 
                     SIPLT FGQGTKLEIK, 
                 
                   and 
                 
                     
                 
                   (SEQ ID NO. 18) 
                 
                   DIVMTQSPDSLAVSLGERATINC KSSQSLLNSSNQKNYLA WYQQKPGQP 
                 
                   PKLLIY FTSTRQS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQHY   
                 
                     SIPLT FGQGTKLEIK. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         4 . The humanized antibody according to  claim 3 , wherein the said humanized antibody contains a heavy chain variable region amino acid sequence comprising or having the sequence selected from a group consisting of SEQ ID NO. 11 and SEQ ID NO. 12; and a light chain variable region amino acid sequence comprising or having the sequence selected from a group consisting of SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 18. 
     
     
         5 . The humanized antibody according to  claim 4 , wherein the said humanized antibody contains a heavy chain variable region amino acid sequence comprising or having the sequence of SEQ ID NO. 12 and a light chain variable region amino acid sequence comprising or having the sequence of SEQ ID NO. 18. 
     
     
         6 . The humanized antibody according to  claim 1 , which has human IgG constant regions of allotype G1m17,1 of the heavy chains and human kappa constant regions of allotype Km3 of the light chains. 
     
     
         7 . A pharmaceutical composition comprising a therapeutically effective amount of a humanized antibody of  claim 1 , which specifically recognizes human CA IX, and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         8 . A method of treatment of a condition comprising the step of administering the pharmaceutical composition of  claim 7  to a subject in need thereof, wherein the condition is selected from the group consisting of cell proliferative disease or disorder, squamous cell carcinoma, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, hodgkin's lymphoma, non-hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, mesothelioma, and head and neck cancer. 
     
     
         9 . The method of treatment of a condition comprising the step of administering the pharmaceutical composition to a subject in need thereof according to  claim 8  wherein the condition is selected from the group consisting of breast cancer, mesothelioma, and glioblastoma expressing CA IX. 
     
     
         10 . A method of treatment of a condition comprising the step of administering the pharmaceutical composition comprising a combination of therapeutically effective amounts of humanized antibodies of according to  claim 1 , which specifically recognize human CA IX, wherein the condition is cell proliferative disease or disorder, and wherein one of the humanized antibodies is administered prior to or subsequently to the administration of a second humanized antibody. 
     
     
         11 . The method of treatment of a condition comprising the step of administering the pharmaceutical composition according to  claim 8  to a subject in need thereof, wherein the daily or weekly dose of the humanized antibody to CA IX ranges from 0.001 mg/kg to 15 mg/kg body weight. 
     
     
         12 . The method of treatment of a condition comprising the step of administering the pharmaceutical composition according to  claim 8  to a subject in need thereof, wherein dosage regimen includes:
 i) multiple, identical or different doses of the humanized antibody; 
 ii) multiple escalating doses of the humanized antibody; or 
 iii) a dose of the humanized antibody once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, or once every 5 weeks. 
 
     
     
         13 . The method of treatment of a condition comprising the step of administering the pharmaceutical composition according to  claim 8  to a subject in need thereof, wherein dosage regiment comprises 1-10 administration cycles, each cycle comprising 2-5 infusions/doses every 1-4 weeks, with a humanized antibody, followed by a break 1-8 weeks between each two cycles. 
     
     
         14 . A diagnostic composition comprising at least one humanized antibody of  claim 1 , and at least one carrier, diluent, or excipient. 
     
     
         15 . A method for diagnosing a cancer in a subject in need thereof, the method comprising contacting a biological sample derived or obtained from said subject with the diagnostic composition of  claim 14 , wherein complex formation beyond a predetermined threshold is indicative of the cancer in said subject. 
     
     
         16 . The method for diagnosing a cancer in a subject according to  claim 15 , wherein the humanized antibody, is linked, bound or conjugated to a paramagnetic, radioactive or fluorogenic moiety that is detectable upon imaging.